Impurities and their limit test

Impurities in Pharmaceuticals and Limit test
Esha Shah 1
Esha Bhavin Shah
Assistant Professor
Department of Pharmaceutical Chemistry
and Quality Assurance
Babaria Institute of Pharmacy
Bits Edu Campus
Email: eshahshah.bip@bitseducampus.ac.in

Outline of the chapter
Definition of impurity
Sources of Impurity
Definition of Assay
Impurities commonly found in
medicinal preparations
Definition of limit test
Importance of Limit test in pharmaceuticals
Limit test for Iron, Arsenic, Chloride, Lead,
Sulphate, Heavy metals
Esha Shah 2

Impure Chemical Compound:
A compound is said to be impure if it is having foreign matter i.e Impurities.
Pure Chemical Compound:
A pure chemical compound refers to that compound which is having no foreign matter i.e
impurities.
Chemical purity means freedom from foreign matter.
Analytically 100 % pure substances are not available
and traces of impurities must be present.
Normally undesirable foreign materials are present in
the pharmaceutical substances.
Esha Shah 3

Impurity means
undesired particles
What is impurity?
Any material that affects the purity of the material of interest.
 Presence of Impurities in the pharmaceutical substances may
produce toxic effects on the body and may also lower down
the active strength of the pharmaceutical substance.
 Impurities commonly in chemical substances include small
quantities of lead, Arsenic, Iron, Chloride and sulphate.Esha Shah 4

Impurities commonly found in medicinal preparations:
 Impurities which have toxic effects on body and bring about unpleasant reactions when
present beyond certain limits. e.g Lead and Arsenic salts.
 The impurities which are able to make substance incompatible with other substances.
 The impurities which if present beyond the limit, affect the storage property of the
pharmaceuticals.
 The impurities which are harmless, but if present beyond the limit, it will lower the
active strength of the medicinal compound. E.g Sodium salt in potassium salt.
 The impurities which may bring about technical difficulties in the use of the substance.
 Impurities such as taste, odour, colour or appearance which can be easily detected by the
senses and make the substance unhygienic and unaesthetic. E.g. Sodium chloride becomes
damp because of the presence of traces of magnesium salts. Also phenolic impurities
present in sodium salicylate alters its odour.
Esha Shah 5

Sources of Impurities in Pharmaceuticals
The type and amount of impurity present in the chemicals or pharmaceutical
substances, depends upon several factors like those listed below:
1) Raw material used in manufacture
2) Reagents used in manufacturing process
3) Method/ process used in manufacture or method of manufacturing
4) Chemical processes used in the manufacture
5) Atmospheric contamination during the manufacturing process
6) Intermediate products in the manufacturing process
7) Defects in the manufacturing process
8) Manufacturing hazards
9) Inadequate Storage conditions
10) Decomposition of the product during storage
11) Accidental substitution or deliberate adulteration with spurious or
useless materials
Esha Shah 6
[GTU important]

1) Raw materials employed in manufacture
• Impurities known to be associated with these chemicals may be carried through the
manufacturing process and contaminate the final product.
• Example
Rock salt-------- Calcium Sulphate (CaSO4) + Magnesium Chloride (MgCl2)= NaCl
prepared
Rock salt contains small amounts of Calcium sulphate and Magnesium chloride.
Thus Sodium chloride prepared from this source will contain traces of Calcium
and Magnesium compounds.
Impurities such as Arsenic, Lead and Heavy metals are present in raw materials
and hence are found in substances. So, it is necessary to use pure chemicals and
substances as raw materials for the manufacturing process.
Calcium sulphate
and Magnesium
chloride
Esha Shah 7

•Example:
 Copper sulphate may be prepared by the action of sulphuric acid on
copper turnings:
Cu+ 2 H2SO4------------------------ CuSO4 + 2 H2O + SO2
Copper turnings are known to have Iron and Arsenic as impurities.
If Large quantities of impurities are present in the raw material
(e.g Copper turnings), they may enter the final product. (CuSO4.5H2O)
Due to this I.P. prescribes limit of tolerance for Arsenic as impurity to be
not more than 8 parts per million in copper sulphate. Similarly it
prescribes a limit of Iron as impurity.
Copper turnings
Esha Shah 8

2) Reagents used in the manufacturing process:
 If reagents used in the manufacturing process are not completely removed by washing,
these may find entry into the final products.
 Example:
Ammoniated mercury may be prepared by adding a solution of Mercuric chloride to dilute
ammonia solution.
HgCl2+ 2NH4OH-------------NH2HgCl + NH4Cl + 2 H2O
Soluble soluble Ammoniated mercury (ppt) (soluble)
The precipitate of Ammoniated mercury (Final Product)contains ammonium hydroxide.
Thus, this precipitate is washed with cold water to remove ammonium hydroxide.
If it is not removed completely by washing with water, the final product may contain in it
Ammonium hydroxide as impurity.
Esha Shah 9

3) Method or the process used in the
manufacture:
 Many drugs and chemicals (usually organic) are manufactured from different raw
materials, by using different methods or processes.
 Some impurities are incorporated into the materials during the manufacturing process.
 The type and amount of impurity present in the drug/ chemical varies.
 In certain drugs , a multiple-step-synthesis procedure is used , which produces
intermediate compounds.
 The purification of intermediates is also important, otherwise the impurities present in
the intermediate will get incorporated in the final product.
 Usually side reactions occur during the synthesis.
 Impurities of the product side reactions also occur in the substances. This may
introduce new impurities due to contamination by reagents and solvents at various
stages of the process as described below:
a) Reagents employed in the process
b) Reagents added to remove other impurities
c) Solvents
d) Action of solvents and reagents on reaction vessels.
Esha Shah 10

C) Solvents:
Water is the cheapest solvent available and has been used wherever possible.
Tap Water It has Ca+2, Mg+2, Na+, Cl-, SO4
-2 and CO3
-2 as
impurities in small amounts
Softened water It is obtained by allowing the tap water to pass
through the sodium form of Zeolite which
removes divalent cations like Ca+2 and Mg+2
from tap water in exchange of sodium.
So, softened water contains Na+, Cl- ions as
impurity.
De-mineralised
water
It is obtained by passing tap water through
columns packed with ion exchange resin.The
water obtained from this process is free from
Ca+2, Mg+2, Na+, Cl-, SO4
-2 and CO3
-2
Thus the final product is free from these
impurities.
The water obtained from this source may still
contain organic impurities and so final product
contains organic impurities.
Distilled water It is considered the best but it is very costly.
Esha Shah 11

a) Reagents employed in the manufacturing process:
• Soluble alkali in Calcium carbonate arises from sodium carbonate used in the
process.
• Calcium carbonate is obtained by interaction of a soluble calcium salt and a
soluble carbonate and therefore the product will contain traces of soluble alkali,
which the washing process has failed to remove.
b) Reagents added to remove other impurities:
• Potassium bromide contains traces of Barium, which is added in the
manufacturing process to remove excess of sulphate.
Esha Shah 12

d) Action of solvents and reagents on reaction vessels:
During manufacturing process, some of the solvents and reagent may undergo reaction with
metals of reaction vessel and may dissolve these metals, which appear as impurities in the
final product.
Example:
 Iron is known to contain Arsenic impurity.
 The inorganic compounds manufactured in Iron vessel will contain Arsenic and Iron as
impurities.
 Thus IP has prescribed limit test for Arsenic and Iron for most inorganic compounds.
Iron vessel
Esha Shah 14

4) Chemical process used in the manufacture:
 For the synthesis of drugs, many chemical reactions such as Nitration, Halogenation,
Oxidation, reduction, hydrolysis are involved.
 In these chemical processes, different chemicals are used.
 Tap water is generally used in the various processes and it is often having Cl-,Mg+2,
Ca+2 ions, which are generally found in the substance which is being manufactured.
Esha Shah 15

5) Atmospheric contamination during the manufacturing
process
 In the industrial areas, the atmosphere is contaminated with dust particles and
some gases like Hydrogen sulphide, Sulphur dioxide, and black smoke.
 During the manufacture or purification of the pharmaceutical products, these
impurities enter the final products.
 There are many pharmaceutical products which when manufactured are
contaminated with atmospheric CO2 and water vapour. E.g NaOH absorbs
atmospheric CO2.
 2NaOH + CO2 -------------------------------- Na2CO3 + H2O
 Due to this reaction, NaOH should not be kept open for a longer time during its
manufacture.
 Therefore, IP has prescribed that Sodium hydroxide should not contain more than
3% of sodium carbonate.
Esha Shah 16

6) Defects in the manufacturing process:
In many manufacturing processes, there are defects like imperfect mixing, incompleteness,
non-adherence to proper temperature, pressure, pH or reaction conditions, which may
give chemical compounds with impurities in them.
Example:
• Zinc oxide may be prepared by heating metallic zinc to bright redness in a current of air.
The vapours of Zinc burn to form Zinc oxide which is collected as a fine white powder.
• But if there is less heat or air or both, zinc metal is not completely converted to zinc
oxide.
• Thus the final product, Zinc oxide may still contain metallic zinc as impurity.
• So, IP has prescribed a test for Zinc metal in zinc oxide.
Esha Shah 17

7) Intermediate products in the manufacturing process:
 There are some intermediates which are produced during the manufacturing process.
Sometimes these intermediates may be carried through to the final product as impurity.
 Example:
Potassium iodide is prepared by reacting Iodine with Potassium hydroxide.
6KOH+ 3I2-------------------- 5KI + KIO3 + 3H2O
The resulting solution is first evaporated and then heated with charcoal.
KIO3 + 3C---------- KI + 3CO
 In this process if the intermediate product (KIO3) is not completely converted into KI,
then it may be carried through to the final product as an impurity.
Esha Shah 18

8) Manufacturing hazards:
Particulate
contamination
Process errors
Cross
contamination
Microbial
contamination
Packing errors
Esha Shah 19

Particulate contamination:
 The presence of unwanted particulate matter can arise due to dirt, dust, glass,
porcelain or plastic fragments from sieves, granulating or tableting machines or
from product containers.
 Ware and tare of equipment or improperly cleaned equipment may also cause
particulate contamination.
 Clarity of solutions for injection is particularly important.
 E.g Metal particles which have been found in eye ointments packed in metal
tubes.
Esha Shah 20

Process errors:
 Gross errors arising from incomplete solution of a solute in a liquid preparation must be
detected readily by the normal analytical control procedures.
 Minor errors arise if the manufacturing tolerance for the quantity of active ingredient in
the product has been wide.
Cross contamination:
 The handling of powders, granules, and tablets in large bulk creates air-borne dust,
which leads to cross contamination of the product.
 So, face masks and special extraction equipment are used to protect operators from
harmful effects of drugs.
 E.g penicillin preparation requires special handling during its manufacture.
Esha Shah 21

Microbial contamination:
• Parenteral preparations and ophthalmic preparations require special care against
microbial contamination.
• Many liquid preparations and creams are liable to bacterial and fungal contamination. So
care should be taken.
• Eg. Acacia, senna, tragacanth---They should be controlled for Salmonellae.
Packing errors:
• Products of similar appearance such as tablets of same size, shape, colour packed in
similar containers can constitute a potential source of danger.
• Improper labelling or destruction of stock of unused labels also constitutes a major
packaging hazard.
Esha Shah 22

9) Storage conditions:
 The chemical substances when prepared have to be stored in different types of containers
depending upon:
 Nature of the material
 Batch size
 Quantity
 Many types of materials are used for storage purpose like plastic, polythene, iron vessels,
stainless steel and aluminium.
 Leaching out effect: Alkalies stored in ordinary glass containers extract lead from it,
which in found as impurity in the final product.
 Strong chemicals react with iron containers and extract Iron an impurity in final product.
Reaction of these
substances
With materials of
storage vessels
The products
formed are
found as
impurities in the
stored materials
Esha Shah 23

Inadequate storage and their effects are as follows:
a) Filth: Stored products may become contaminated with dust, bodies of insects, animal and
insect excreta.
b) Chemical instability: decomposition because of light, traces of acid or alkali, air
oxidation, water vapour, CO2 and traces of metallic ions.
e.g light sensitive materials should be stored in amber colored bottles.
c) Reactions with container materials: e.g salicylic acid ointment must not be stored in
metal tubes.
d) Physical changes: The occurance of changes in the physical form of drug like change in
crystal size can lead to change in efficiency of product.
e) Temperature effect: Chemical and physical changes occur if materials are not stored at
proper temperature.
Esha Shah 24

10) Decomposition of the product during storage:
 Chemical decomposition, analysis or breakdown is the separation of a chemical
compound into elements or simpler compounds. It is sometimes defined as the exact
opposite of a chemical synthesis. Chemical decomposition is often an undesired chemical
reaction.
 Some substances decompose on storing due to presence of air, light and oxygen. So,
the final product is contaminated.
 Deliquescent substances, absorb water from the atmosphere and get liquefied.
 Decomposition products appear as impurities in the substances.
Esha Shah 25

11) Accidental substitution or deliberate adulteration with spurious or
useless materials:
 It is possible to avoid accidental substitution by storing the toxic substances together
separately or in a locked cupboard.
 Many pharmaceutical chemicals are adulterated with cheaper substances.
 E.g The expensive potassium may be adulterated with sodium bromide.
Esha Shah 26

Effect of Impurities:
The impurities present in the substances may give following effects:
 Impurities having toxic effects may be injurious to health, if present above
certain limits.
 Traces of impurities, may exert a cumulative toxic effect after a certain time.
 Impurities may lower the active strength of the substance.
 Impurity may decrease shelf life of substance.
 Impurity may cause incompatibility with other substances.
 Impurities may cause a physical or chemical change in the properties of the
substance, so making the substance medicinally useless.
 May cause change in color, odour and taste.
Esha Shah 27

Test for purity:
 Pharmacopoeia prescribes the “Test for purity” for pharmaceutical substances to
check their freedom from undesirable impurities.
 Pharmacopoeia will decide and fix the limit of tolerance for these impurities.
 For certain common impurities for which pharmacopoeia prescribes the test of
purity are:
 Colour, odour, taste
 Physicochemical constants (Iodine value, saponification value, melting point,
refractive index etc.)
 Acidity, alkalinity, pH
 Humidity (Estimation of moisture)
 Cations and anions
 Ash
 Arsenic or lead
 Loss on drying
 Loss on ignition
Esha Shah 28

Impurity means
undesired particles
What is impurity?
Any material that affects the purity of the material of interest.
 Presence of Impurities in the pharmaceutical substances may
produce toxic effects on the body and may also lower down
the active strength of the pharmaceutical substance.
 Impurities commonly in chemical substances include small
quantities of lead, Arsenic, Iron, Chloride and sulphate.Esha Shah 29

Limit tests:
 Tests being used to identify the impurity.
 Tests being used to control the impurity.
 Definition: Limit tests are quantitative or semi quantitative test
designed to identify and control small quantities of impurities which
are likely to be present in the substances.
Esha Shah 30

Factors affecting limit tests:
 Specificity of the tests
 Sensitivity
 Control of personal errors (Analyst errors)
• Test in which there is no visible reaction
• Comparison methods
• Quantitative determination
Esha Shah 31

Types:
 Tests in which there is no visible reaction
 Comparison methods
 Quantitative determinations
Esha Shah 32

Turbidity means cloudiness or haziness.
Esha Shah 33

Limit test for IRON:
 Limit test of Iron is based on the reaction of iron in ammonical solution with
thioglycollic acid in presence of citric acid to form iron thioglycolate (Ferrous
thioglycolate complex) which produces pale pink to deep reddish purple color in alkaline
media.
 Thioglycolic acid is used as reducing agent.
 The color of the Ferrous thioglycolate complex fades in the presence of air due to
oxidation.
 Also, the color is destroyed in presence of oxidizing agents and strong alkalis.
 The purple color is developed only in alkaline media. So ammonia solution is used.
 But ammonia reacts with iron, forms precipitate of ferrous hydroxide.
 Thus citric acid is used which prevents the precipitate of iron with Ammonia by forming
a complex with iron as iron citrate.
Esha Shah 34

Test sample Standard compound
Sample is dissolved in specific amount
of water and then volume is made up to
40 ml
2 ml of standard solution of iron diluted
with water upto 40 ml
Add 2 ml of 20 % w/v of citric acid
(iron free)
Add 2 ml of 20 % w/v of citric acid
(iron free)
Add 2 drops of thioglycollic acid Add 2 drops of thioglycollic acid
Add ammonia to make the solution
alkaline and adjust the volume to 50 ml
Add ammonia to make the solution
alkaline and adjust the volume to 50 ml
Keep aside for 5 min Keep aside for 5 min
Color developed is viewed vertically
and compared with standard solution
Color developed is viewed vertically and
compared with standard solution
Procedure:
Note: All the reagents used in the limit test for Iron should themselves be iron free.
Esha Shah 35

Observation:
The purple color produce in sample solution should not be greater than standard solution. If
purple color produces in sample solution is less than the standard solution, the sample will
pass the limit test of iron and vice versa.
Reasons:
 Citric acid forms complex with metal cation and helps precipitation of iron by
ammonia by forming a complex with it.
 Thioglycolic acid helps to oxidize iron (II) to iron (III).
 Ammonia is added to make solution alkaline. The pale pink color is visible only
in the alkaline media. The color is not visible in acidic media as ferrous
thioglycolate complex decomposes in high acidic media.
Esha Shah 36

Limit test for Chloride:
The test is used to limit the amount of Chloride as an impurity
in inorganic substances.
Esha Shah 37

Limit test for CHLORIDE:
Principle:
Limit test of chloride is based on the reaction of soluble chloride with silver nitrate in
presence of dilute nitric acid to form silver chloride, which appears as solid particles
(Opalescence) in the solution.
Cl- + AgNO3 -------------------------- AgCl + NO3
-
Soluble
chloride
present as
impurity
 The silver chloride produced in the presence of dilute Nitric acid makes the test solution
turbid, the extent of turbidity depending upon the amount of Chloride present in the
substance is compared with the standard opalescence produced by the addition of Silver
nitrate to a standard solution having a known amount of chloride and the same volume of
dilute nitric acid as used in the test solution.
Esha Shah 38
silver chloride

Specific weight of compound
is dissolved in water
or solution is prepared as
directed in the pharmacopoeia
and transferred in Nessler
cylinder
Take 1 ml of 0.05845 % W/V
solution of sodium chloride in
Nessler cylinder
Add 1 ml of nitric acid Add 1 ml of nitric acid
Dilute to 50 ml in Nessler cylinderDilute to 50 ml in Nessler cylinder
Add 1 ml of AgNO3 solution Add 1 ml of AgNO3 solution
Observe the
Opalescence/Turbidity
Observe the Opalescence/Turbidity
Esha Shah 40

Observation:
The opalescence produce in sample solution should not be greater than standard
solution. If opalescence produces in sample solution is less than the standard
solution, the sample will pass the limit test of chloride and visa versa.
Reasons:
Nitric acid is added in the limit test of chloride to make
solution acidic and helps silver chloride precipitate to
make solution turbid at the end of process as Dilute HNO3
is insoluble in AgCl.
Esha Shah 41

 Pharmacopoeia does not prescribe any numerical value of limit test for chlorides,
sulphate and iron because limit test is based on the simple comparison of
opalescence or colour between the test and standard solution prescribed according
to pharmacopoeia.
 In this type of limit test, the extent of turbidity or opalescence or colour produced
in influenced by the presence of other impurities present in the substance and also
by variation in time and method of performance of test.
 Thus the pharmacopoeia does not prescribe any numerical value of the limit test.
Pharmacopoeia not prescribe any numerical value for limit test for chlorides,
sulphate and iron because limit test is based on simple comparison of opalescence or
color between test and standard solution prescribed according to pharmacopoeia.
The variation in the permissible limits for various substances is obtained by taking
varying quantities of the substances under test. In this type of limit test, the extent of
opalescence or turbidity or color produced is influenced by the presence of other
impurities present in the substance and also by variation in time and method of
performance of tests and hence the pharmacopoeia do not prescribe any numerical
values for the limit test in these test.
Esha Shah 42

 The limit test involve simple comparisons of opalescence, turbidity, or colour with
standard.
 These are semi-qualitative reactions in which extent of impurities present can be estimated
by comparing visible reaction response of the test and standard.
 By this way, extent of reaction is readily determined by direct comparison of test solution
with standard. So pharmacopoeia prefers comparison methods.
Esha Shah 43

Limit test for sulphate:
The Sulfate Limit Test is designed to determine the allowable limit of sulfate contained in a
sample.
Principle:
Limit test of sulphate is based on the reaction of soluble sulphate with barium chloride in
presence of dilute hydrochloric acid to form barium sulphate which appears as solid
particles (turbidity) in the solution.
Then comparison of turbidity is done with a standard turbidity obtained from a known
amount of Sulphate and same volume of dilute Hydrochloric acid have been added to both
solutions.
The barium chloride test solution in the IP has been replaced by Barium sulphate reagent
which is having barium chloride, sulphate free alcohol and a solution of potassium
sulphate. Potassium sulphate has been added to increase the sensitivity of the test.
SO4
-2 + BaCl2 --------------------------- BaSO4 + 2Cl-
Esha Shah 44

Specific weight of compound is
dissolved in water or solution is
prepared as directed in the
pharmacopoeia and transferred in
Nessler cylinder
Take 1 ml of 0.1089 % W/V solution of potassium
sulphate in Nessler cylinder
Add 2 ml of dilute hydrochloric acid Add 2 ml of dilute hydrochloric acid
Dilute to 45 ml in Nessler cylinder Dilute to 45 ml in Nessler cylinder
Add 5 ml of barium sulphate reagent Add 5 ml of barium sulphate reagent
Observe the Turbidity Observe the Turbidity
Procedure:
Esha Shah 45

Barium sulphate reagent contains barium chloride, sulphate free alcohol and small amount
of potassium sulphate.
Observation:
The turbidity produce in sample solution should not be greater than standard solution. If
turbidity produces in sample solution is less than the standard solution, the sample will pass
the limit test of sulphate and vice versa.
Reasons:
Hydrochloric acid helps to make solution acidic.
Potassium sulphate is used to increase the sensitivity of the test by giving ionic
concentration in the reagent.
Alcohol helps to prevent super saturation and so produces a more uniform opalescence.
Esha Shah 46

Limit test for Arsenic:
 Arsenic is a well known undesirable and harmful impurity which is present in medicinal
substances.
 All pharmacopoeias prescribe a limit test for it.
 Pharmacopoeial method is based on the Gutzeit test.
 All the special reagents used in the limit test for Arsenic are marked and distinguished by
letter ‘As T’, which means that they all should be Arsenic free and should themselves
conform to the test for Arsenic.
Esha Shah 47

Principle:
Limit test of Arsenic is based on the reaction of arsenic gas with hydrogen ion to form
yellow stain on mercuric chloride paper in presence of reducing agents like
potassium iodide. It is also called as Gutzeit test and requires special apparatus.
 Arsenic, present as arsenic acid (H3AsO4) in the sample is reduced to arsenious
acid (H3AsO3) by reducing agents like potassium iodide, stannous acid, zinc,
hydrochloric acid, etc. Arsenious acid is further reduced to arsine (gas) (AsH3)
by hydrogen and reacts with mercuric chloride paper to give a yellow stain.
 Substance + dil HCl ------------------------- H3AsO4
(contains Arsenic impurity) Arsenic acid
 H3AsO4 + H2SnO2 -------------------→ H3AsO3 + H2SnO3
Arsenic acid Arsenious acid
H3AsO3 + 6[H] ---------------------→ AsH3 + 3H2O
Arsenious acid nascent hydrogen Arsine gas
The depth of yellow stain on mercuric chloride paper will depend upon the
quantity of arsenic present in the sample.Esha Shah 48

When the sample is dissolved in acid, the Arsenic present in the sample
gets converted to Arsenic acid.
By action of reducing agents like Potassium iodide, stannous acid etc.,
Arsenic acid gets reduced to arsenious acid.
The nascent hydrogen formed during the reaction, further reduces
Arsenious acid to Arsine gas, which reacts with mercuric chloride paper,
giving a yellow stain.
Esha Shah 49

Apparatus:
 It is having a wide mouthed glass bottle of 120 mL capacity having mouth of about
2.5 cm in diameter. This bottle is fitted with a rubber bung through which passes a glass
tube, 20 cm long.
 External diameter=0.8 cm
 Internal diameter=0.65 cm
 The tube is constricted at its lower end extremity to about 1 mm diameter and there is
blown a hole, not less than 2 mm in diameter, in the side of the tube near the constricted
part.
 The upper end of the glass tube is fitted with two rubber bungs(25 mm x 25 mm), each
having a hole bored centrally and exactly 6.5 mm in diameter.
 One of the bungs has been fitted to the upper end of the tube, while the second bung has
to be fitted upon the first bung in such a way that the mercuric chloride paper gets
exactly sandwiched between the central perforation of the two.
 The bungs are kept in close contact by using rubber band or spring clip in such a manner
that the gas evolved from the bottle must have to pass through the 0.65 mm internal
circle of mercuric chloride paper.
 During the test, the evolved gases have been passing through the side hole, the lower
hole serving as an exit for water which condenses in the constricted part of the tube.
 An important feature has been the standardization of the area of Mercuric chloride paper
which is exposed to the reaction of arsine gas.
Esha Shah 51

The test solution is prepared by
dissolving specific amount in water
and stannated HCl (arsenic free) and
kept in a wide mouthed bottle.
A known quantity of dilute arsenic
solution in water and stannated HCl
(arsenic free) is kept in wide mouthed
bottle.
1 g of KI 1 g of KI
5 ml of stannous chloride acid solution 5 ml of stannous chloride acid solution
10 g of granulated zinc is added (all
this reagents must be arsenic free).
Keep the solution aside for 40 min
10 g of zinc is added (all this reagents
must be arsenic free).
Keep the solution aside for 40 min
Stain obtained on mercuric chloride paper is compared with standard solution.
Standard stain must be freshly prepared as it fades on keeping.
Inference: If the stain produced by the test is not deeper than the standard
stain, then sample complies with the limit test for Arsenic.
Esha Shah 52

Reasons:
Stannous chloride is used for complete evolution of arsine.
Zinc, potassium iodide and stannous chloride is used as a reducing agent.
Hydrochloride acid is used to make the solution acidic
Lead acetate paper are used to trap any hydrogen sulphide which may be evolved
along with arsine.
Esha Shah 53

Use of stannated Hydrochloric acid:
If pure zinc and HCl are used, the steady evolution of gas does not occur. This produces
improper stain (e.g slow evolution produces short but intense stain while rapid evolution of
gas produces long but diffused stain.)
So, to get steady evolution of gas, stannated hydrochloric acid is used.
Use of Lead Acetate solution:
H2S gas may be formed during the experiment as zinc contains sulphides as impurities. It
gives black stain to HgCl2 paper and so will interfere the test.
Hence, gases evolved are passed through cotton wool plug moistened with lead acetate,
where H2S gas is trapped as PbS.
Use of Potassium iodide:
KI is converted to HI which brings about reduction of unreacted pentavalent arsenic to
trivalent Arsenic. Thus, reproducible results can be obtained. If it is not used then some
pentavalent Arsenic may remain unreacted.
Esha Shah 54

Limit test for heavy metals
 The limit test for heavy metals is designed to determine the content of metallic impurities
that are coloured by hydrogen sulphide or sodium sulphide under the condition of the test
should not exceed the heavy metal limits given under the individual monograph.
 The heavy metals (metallic impurities) may be iron, copper, lead, nickel, cobalt, bismuth,
antimony etc.. The limit for heavy metals is indicated in the individual monograph in
term of ppm of lead i.e. the parts of lead per million parts of the substance being
examined
 In substances the proportion of any such impurity (Heavy metals) has been expressed as
the quantity of lead required to produce a color of equal depth as in a standard comparison
solution having a definite quantity of lead nitrate.
 The quantity is stated as the heavy metal limit and is expressed as parts of lead (by
weight) per million parts of the test substance.
 The limit test for heavy metals has been based upon the reaction of the metal ion
with hydrogen sulphide, under the prescribed conditions of the test causing the
formation of metal sulphides.
 These remain distributed in colloidal state, and give rise to a brownish coloration.
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 I.P limit for heavy metals in 20 ppm.
 The test solution is compared with a standard prepared using a lead solution (as the heavy
metal).The metallic impurities in substance are expressed as parts of lead per million parts
of substance.
 IP has adopted 3 methods for this:
 Method I: The method is applicable for the samples which give clear colourless solutions
under specified conditions of test.
 Method II: The method is applicable for the samples which DO NOT give clear
colourless solutions under specified conditions of test.
 Method III: Used for substances which give clear colourless solutions in sodium
hydroxide medium.

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Limit test for lead:
Lead is a most undesirable impurity in medical compounds
and comes through use of sulphuric acid, lead lined
apparatus and glass bottles use for storage of chemicals.
Principle:
Limit test of lead is based on the reaction of lead and diphenyl
thiocabazone (dithizone) in alkaline solution to form lead
dithizone complex which is red in color.

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 Dithizone in chloroform, is able to extract lead from alkaline aqueous solutions as
a lead dithizone complex (Red in colour)
 The original dithizone is having a green colour in chloroform while the lead-
dithizone is having a violet color.So, resulting color at the end of the process is
read.
 The intensity of the color of complex is dependant upon the amount of lead in
the solution.
 The color of the lead-dithizone complex in chloroform has been compared with a
standard volume of lead solution, treated in the same manner.
 In this method, the lead present as an impurity in the substances, gets separated by
extracting an alkaline solution with a dithizone extraction solution.
 The interference and influence of the other metal ions has been eliminated by
adjusting the optimum pH for the extraction by employing Ammonium
citrate/ potassium cyanide.

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Method:
• Sample solution is transferred to a separating funnel.
• To it 6 ml of ammonium citrate, 2 ml potassium cyanide and 2 ml of hydroxalamine HCl
are added.
• 2 drops of phenol red
• Solution is made alkaline by adding ammonia solution.
• This is then extracted with 5 ml portions of dithizone solution until it becomes green.
• The combined dithizone extracts are shaken for 30 seconds with 30 ml of nitric acid and
chloroform layer is discarded.
• To the acid solution 5 ml of standard dithizone solution is added and 4 ml ammonium
cyanide and solution is shaken for 30 sec.
• Similarly prepare standard.

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 Observation:
The intensity of the color of complex, is depends on the amount of lead
in the solution. The color produced in sample solution should not be
greater than standard solution. If color produces in sample solution is less
than the standard solution, the sample will pass the limit test of lead and
vice versa.
Reasons:
 Ammonium citrate, potassium cyanide, hydroxylamine
hydrochloride is used to make pH optimum so interference and
influence of other impurities have been eliminated.
 Phenol red is used as indicator to develop the color at the end of process
Lead present as an impurities in the substance, gets separated by
extracting an alkaline solution with a dithizone extraction solution.

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A known quantity of sample solution is
transferred in a separating funnel
A standard lead solution is prepared equivalent to
the amount of lead permitted in the sample under
examination
Add 6ml of ammonium citrate Add 6ml of ammonium citrate
Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride
Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride
Add 2 drops of phenol red Add 2 drops of phenol red
Make solution alkaline by adding ammonia solution. Make solution alkaline by adding ammonia solution.
Extract with 5 ml of dithizone until it becomes
green
Extract with 5 ml of dithizone until it becomes green
Combine dithizone extracts are shaken for 30 mins
with 30 ml of nitric acid and the chloroform layer is
discarded
Combine dithizone extracts are shaken for 30 mins
with 30 ml of nitric acid and the chloroform layer is
discarded
To the acid solution add 5 ml of standard dithizone
solution
To the acid solution add 5 ml of standard dithizone
solution
Add 4 ml of ammonium cyanide Add 4 ml of ammonium cyanide
Shake for 30 mins Shake for 30 mins
Observe the color Observe the color

Aq. Ammonia is added in limit test of lead:
Pb+ S-------------PbS
In limit test of lead, PbS is produced by addition of standard H2S , to the
solution containing lead.
pH 3-4 is necessary for the precipitation of PbS. So aq. Ammonia /
acetic acid is added to maintain that pH.
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Modified limit test for Chlorides
Depending upon the nature of the substance,
some modifications have to be adopted for the
preparation of the solution.
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(a) Alkaline substances have to be dissolved in acid so that effervescence ceases and
much of the free acid is left in the solution as is prescribed in the test.
(b) Insoluble substances are generally extracted with water and then filtered, and the
filtrate is used for the test, because the presence of insoluble substance modifies the
opalescence and colour.
(c) Salts of organic acids like sodium benzoate, sodium salicylate, etc. liberate free
water insoluble organic acid during acidification which is filtered off and the filtrate is
employed for the test.
(d) Coloured substances like crystal violet, malachite green, etc. are carbonised and the
ash so produced is extracted in water.
(e) Deeply coloured substances have to be decolourised before test e.g., potassium
permanganate is reduced by boiling with alcohol and the filtrate is used.
(f) Reducing substances like hypophosphorus acid, which react with silver nitrate in the
limit test for chlorides should be oxidized with nitric acid or some other oxidizing
agents before carrying out the test.
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Aim To perform the limit test for chloride in potassium permanganate sample (according to
IP’96)
Requirement:
Nessler’s cylinder, measuring cylinder, pipette, spatula, distilled water, dilute nitric acid, 0.1 M
silver nitrate solution, potassium permanganate sample
Principle:
The limit test for chloride based on the reaction between soluble chloride impurities present in the
substance and silver nitrate solution to give white precipitates of silver chloride. These white
precipitates are insoluble in dilute nitric acid and hence give turbidity or opalescence to the test
solution. The extent of the turbidity produced depends upon the amount of the chloride present in
the substance which is compared with a standard opalescence produce by addition of silver
nitrate to a standard solution having known amount of chloride and the same volume of the dilute
nitric acid as the use in the test solution. If the turbidity developed in the sample is less than the
standard turbidity, the sample passes the limit test for chloride and vice-versa. As potassium
permanganate gives purple color aqueous solution that interferes in the comparison of
opalescence or turbidity, therefore the aqueous solution must first be decolorized.
Potassium permanganate is oxidizing agent while ethanol is reducing agent.
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When potassium permanganate solution is treated with ethanol in presence of heat
the redox reaction will take place, i.e. potassium permanganate gets reduced to
manganese dioxide (precipitates). The filtrate of the reaction is colorless that is
subjected to proceed for limit test for chloride.
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Chemical Reaction:
2 KMnO4 + 3 C2H5OH---------- 2 MnO2 + 2 KOH + 2 CH3CHO + 2 H2O
Sr.
No.
STANDARD SOLUTION SR.
NO
TEST SOLUTION
1 Take 10 ml chloride standard solution (25 ppm
chloride) and add 5 ml water in a Nessler’s
cylinder.
1 Transfer the prepared test solution in Nessler’s
cylinder
2 Add 10 ml of dilute nitric acid and dilute to 50 ml
with distilled water
2 Add 10 ml of dilute nitric acid and dilute to 50
ml with distilled water
3 Add 1ml of 0.1 M silver nitrate solution and stir
immediately with glass rod and allow standing
for 5 minutes protected from light.
3 Add 1ml of 0.1 M silver nitrate solution and
stir immediately with glass rod and allow
standing for 5 minutes protected from light.
Compare the turbidity or opalescence produced in test solution with respect to
standard solution and report the result and conclusion.

Observation and conclusion:
Observation and conclusion will be of two types:
If the intensity of turbidity or opalescence appears to be more in test
solution than the standard solution then conclusion is impurities of
chloride in given sample is over the limit as per IP’96. Hence, sample
do not passes the limit test for chlorides.
If the intensity turbidity or opalescence appears to be less or equal in
test solution than the standard solution then conclusion is impurities
of chloride in given sample is under the limit as per IP’96. Hence,
sample passes the limit test for chloride
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To perform the limit test for sulphate in Potassium
permanganate sample (According to IP’96)
Principle:
It is a comparison method. It involves the comparison of opalescence or turbidity of test sample verses
standard sample which contain the definite amount of sulphate impurities.
The limit test of sulphate is performed on the basis of reaction between the barium chloride reagent
(containing barium chloride, sulphate free alcohol and solution of potassium sulphate (K2SO4) and
soluble sulphate in the sample with formation of barium sulphate (BaSO4) white precipitates.
Sulphate free alcoholic potassium sulphate is added to increase the sensitivity of the test. Very small
amount of barium sulphate present in the reagents acts as a seeding agents for precipitation of barium
sulphate, if sulphate is present in the sample under the test.
Ethanol is added to prevent the super saturation i.e. the crystallization of sulphate with any other ion.
As potassium permanganate gives purple colored aqueous solution that interferes in the comparison of
opalescence or turbidity, therefore it requires to be decolorized. Potassium permanganate is oxidizing
agent while ethanol is reducing agent. When potassium permanganate solution is treated with ethanol
in presence of heat the redox reaction takes place, i.e. potassium permanganate gets reduced to
manganese dioxide (precipitates) and ethanol gets oxidized to form ethanal. The filtrate of the reaction
is colorless that is subjected to proceed for limit test for sulphate.
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Sr.
No.
STANDARD SOLUTION SR.
NO
TEST SOLUTION
1 Take 1 ml 25% w/v barium chloride in Nessler’s
cylinder and add 1.5 ml of ethanolic sulphate
standard solution (10 ppm SO4
-2). Mix and allow to
stand for 1 minutes
1 Take 1 ml 25% w/v barium chloride in Nessler’s
cylinder and add 1.5 ml of ethanolic sulphate
standard solution (10 ppm SO4
-2). Mix and allow to
stand for 1 minutes
2 Add 15 ml of standard sulphate solution (10 ppm
SO4
-2) and 0.15 ml of 5M acetic acid.
2 Transfer prepared test solution and add 0.15 ml of
5 M acetic acid.
3 Add sufficient distilled water to produced 50 ml.
Stirred it immediately and allow standing for 5
minutes.
3 Add sufficient distilled water to produced 50 ml.
Stirred it immediately and allow standing for 5
minutes.
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Reaction:
2 KMnO4 + 3 C2H5OH -------------------- 2 MnO2 + 2 KOH + 2 CH3CHO + 2 H2O

• Compare the turbidity or opalescence in the
test solution by viewing transversely both
solutions against black background.
•
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Impurities and their limit test

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